Publication protocol
For each sample, digestion and ligation reactions were carried out simultaneously at 37°C for 3 hours on 50 ng of genomic DNA, using 2 units of restriction enzyme Bsp1286I (New England Biolabs, NEB), 80 units of T4 DNA ligase (NEB) and 0.05 μM Bsp1286I adaptors I (Table (Table1),1), in a buffer with final concentrations of 10 mM Tris-OAc, 50 mM KOAc, 10 mM Mg(OAc)2, 5 mM DTT (pH 7.8), 1 mM ATP and 100 ng/ml Bovine Serum Albumin (NEB). The obtained ligated products served as template in a first round of PCR amplification. For this purpose, ligated products were diluted five times with sterile water and 2.5 μl of the diluted product were added to a 22.5-μl PCR reaction mix leading to final concentrations of 0.04 μM of Bsp1286I primers I (Table (Table1),1), 0.4 μM of PonyB primers (Table (Table1),1), 10 mM Tris-Cl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.1 mM of each dNTP and 1 unit of RedTaq DNA Polymerase (Sigma). The amplification reaction was performed with the following conditions: 94°C for 1 min; 20 cycles of 94°C for 30 sec, 50°C for 40 sec and 72°C for 1 min; followed by a final 7-min extension step at 72°C. The resulting PCR product was diluted 5 times with sterile water and 2.5 μl of the diluted product served as a template for a second round of amplification performed exactly as the first round except that the final volume was 50 μl, the final concentration of both primers and of each dNTP was 0.2 μM and 0.05 mM, respectively, and 2 units of RedTaq DNA polymerase were used.
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